CN115089617A - Application of nocardia rubra cell wall skeleton in treatment of chronic cervicitis - Google Patents

Abstract

The present disclosure relates to the use of nocardia rubra cell wall scaffolds in the treatment of chronic cervicitis. In particular, the present disclosure provides nocardia rubra cell wall skeleton for use in preventing or treating chronic cervicitis with an infection by a high risk HPV selected from any one of or a combination of: 16. models 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68.

Description

Application of nocardia rubra cell wall skeleton in treatment of chronic cervicitis

Technical Field

The present disclosure relates to the fields of medicine and biopharmaceutical technology. In particular, the disclosure relates to the use of nocardia rubra cell wall scaffolds in the treatment of chronic cervicitis.

Background

Chronic cervicitis is the most common disease in women of childbearing age, and is often caused by streptococcus, enterococcus, escherichia coli and staphylococcus, and special pathogenic microorganisms comprise chlamydia trachomatis and gonococcus. In addition, childbirth and mechanical injury are also the causative factors of chronic cervicitis.

Nocardia rubra (Nocardia rubra) is one of Nocardia. The red nocardia thallus is fermented, cell crushed and degraded by protease to obtain the red nocardia cell wall skeleton.

In the prior art, the nocardia rubra cell wall skeleton may be commercially available, in particular, from the Liaoning Grace Shite biopharmaceutical Co., Ltd. Nocardia rubra cell wall skeleton has been used for the treatment of pre-cervical lesions (CN101073583A), anti-human papilloma virus (CN1935262A), skin lesions or ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster (CN 1879661A).

Disclosure of Invention

In a first aspect, the present disclosure provides a nocardia rubra cell wall skeleton.

Nocardia rubra refers to the genus Nocardia, the species Nocardia rubra (Nocardia rubra).

Identification of nocardia rubra: one skilled in the art can taxonomically identify a strain of bacteria according to known or future microbial identification techniques, e.g., available identification techniques include morphological, physiobiochemical features, 16S rRNA, etc. The skilled person understands that as technology advances, identification techniques involve different means, and in earlier times morphological and biochemical identification methods were mainly used, but the reliability of such methods is not high. After the advent of sequencing technology, the skilled artisan can identify strains in a more reliable manner. For example, when the DNA sequence of 16S rRNA is identified as having 97% (inclusive) or more similarity, two genera are judged to be of the same species. For nocardia rubra, known strains deposited in the international (or national grade) collection of species are used as model strains and compared therewith.

In the present disclosure, "nocardia rubra cell wall" can be understood as both an intact cell wall and an incomplete cell wall (e.g., disrupted, or partially degraded). The skilled artisan, in light of the present disclosure, will appreciate that the component exhibiting the desired activity is derived from the cell wall of nocardia rubra (e.g., is the cell wall itself or a constituent thereof). Therefore, various forms of intact cell walls, disrupted cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, etc., which are allowed to be used in clinical applications, are included in the scope of the present disclosure.

The cell wall skeleton of the present disclosure is not to be understood as merely representing a cross-linked network entity within the cell wall, and the skilled person will understand that the term does not exclude other cell wall components adsorbed, bound, carried on the cross-linked network entity.

In a specific example, the cell wall skeleton of the present disclosure is the product of bacteria after disruption and decontamination (protein, nucleic acid, cell membrane, lipid).

In a specific embodiment, the cell wall scaffold is nocardia rubra cell wall scaffold corresponding to the national drug standard S20030009 or equivalent thereof.

The skilled artisan will appreciate that S20030009 is an administrative license initial number issued by the drug administration, which number varies with the renewal of the certificate, the law, and the adjustment of numbering rules. However, the product standard (product parameters and/or quality requirements) represented by the number is not changed regardless of the change in the number. Thus, S20030009 in the present disclosure should be understood to also include: s20030009 itself, cell wall scaffolds corresponding to the same number and update number thereof, also includes cell wall scaffolds having the same product parameters and/or quality requirements as S20030009.

"cell wall skeleton having the same product parameters and/or quality requirements" or "cell wall skeleton corresponding to the same standard" means that the cell wall skeleton to be tested and the cell wall skeleton shown in S20030009 have active ingredients (muramic acid, sugar, lipid) that have no statistically significant difference under the same test method.

In other embodiments, the nocardia rubra cell wall skeleton is obtained by a method comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) removing lipids from the disruption product;

3.2) removing nucleic acids from the disruption product;

3.3) removing proteins from the disruption product;

3.4) removing cell membranes from the disruption product;

3.5) obtaining a red nocardia cell wall skeleton;

4) optionally, subpackaging;

5) optionally, freeze-drying the nocardia rubra cell wall skeleton;

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel, and steps 4) and 5) can be interchanged.

For disruption of nocardia rubra, the aim is to remove intracellular material. Therefore, the techniques of ultrasonic crushing, high-pressure homogenizer crushing, lysozyme and the like can be adopted. The skilled artisan will appreciate that any known or future method suitable for disrupting gram-positive bacteria is suitable for use in the presently disclosed embodiments.

The skilled person has the ability to adapt the specific parameters and equipment of culturing, disruption, separation, collection, removal of impurities, packaging in response to the subsequent application (e.g. topical application) of the active ingredient (cell wall and its constituent components) in order to avoid introduction of factors in the preparation step that affect the subsequent application.

In some embodiments, the lipids in the disrupted product are removed using an organic solvent. In some embodiments, the DNA and RNA in the disruption products are removed using a nuclease. In some embodiments, the protein in the disruption product is degraded using a hydrolase. In some embodiments, the cell membranes in the disruption products are removed using a surfactant.

In some embodiments, the average particle size of the disruption is from 10nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ± 10nm, and ranges between any two of the foregoing values. The methods of particle size testing are well known in the art.

In some specific embodiments, the average particle size of the disruption is from 10nm to 800 nm.

In other specific embodiments, the average particle size of the disruption is from 10nm to 500 nm.

In specific embodiments, the dispensing means into a bottle or ampoule. Just prior to use, a solvent (e.g., sterile water) is added to the vial or ampoule. As an example, the bottle is a vial (visual, made of borosilicate glass or soda lime glass).

In a second aspect, the present disclosure provides the use of the aforementioned nocardia rubra cell wall scaffold in the preparation of a medicament for preventing or treating chronic cervicitis with high risk HPV infection in a subject.

The high risk HPV is selected from any one or combination of the following: 16. models 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68.

The chronic cervicitis is selected from any one or a combination of the following: cervical metaepithelial dislocation, cervical polyps, cervical mucositis, cervical gland cysts, cervical hypertrophy (classifications are found, for example, in the publication of the national public health press, seventh edition).

In some embodiments, ectopic cervical columnar epithelium is the most common local feature in the inflammatory processes of chronic cervicitis. The surface of the cervix presents red lesions, which are the result of the exfoliation of the squamous epithelium, which is replaced by columnar epithelium, and the appearance of the sub-epithelial vessels. It is often clinically classified into light I (lesion area less than total cervical area 1/3), medium II (1/3-1/2), and severe III (more than 1/2) according to lesion area.

In some embodiments, cervical polyps are one of the manifestations of chronic cervicitis. The chronic inflammation causes the local mucous membrane of the cervical canal to be proliferated due to long-term stimulation, and the proliferated mucous membrane gradually protrudes from the base part to the external cervical orifice to form polyp due to the tendency of removing foreign matters from the uterus.

In some embodiments, cervical mucositis is one of the common manifestations of chronic cervicitis. Lesions are located in the mucosa of the cervical canal and submucosal tissues.

In some embodiments, a cervical gland cyst is one of the common manifestations of chronic cervicitis. In the cervical columnar epithelial ectopic recovery process, the neogenetic squamous epithelium covers the cervical gland orifice or extends into the gland duct to block the gland orifice; the connective tissue hyperplasia or scar around the glandular duct forms to press the glandular duct, so that the glandular duct is narrowed or even blocked, the drainage of gland secretion is blocked, and the cyst formed by retention is called cervical Leng's cyst. A cyst of a cervical gland is an inflammation, not a tumor.

In some embodiments, cervical hypertrophy is one of chronic cervicitis. Inflammatory changes caused by infection of the cervical mucosa by pathogens. Hypertrophic cervical surfaces also exhibit squamous epithelial detachment and columnar epithelial hyperplasia due to injury or inflammatory irritation.

In some embodiments, the medicament is prepared in a dosage form selected from any one of: injection, unguent, cream, lotion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, membrane, patch, and suppository.

In a third aspect, the present disclosure provides a pharmaceutical composition or medicament for preventing or treating chronic cervicitis with high risk HPV infection in a subject, comprising: a pharmaceutically acceptable carrier and the nocardia rubra cell wall skeleton of the present disclosure.

The pharmaceutical composition or medicament of the present disclosure may be prepared in the form of a unit dose (or unit formulation).

In some embodiments, the pharmaceutical composition or medicament may be prepared in a liquid state (liquid formulation).

In other embodiments, the pharmaceutical composition or medicament may be prepared as a solid (dry powder formulation or lyophilized powder formulation).

The skilled person understands that liquid formulations and dry powder formulations (or lyophilized powder formulations), which can be interconverted, differ only in the water content. Removing most or all of water in the liquid preparation to obtain dry powder preparation (or lyophilized powder preparation). Dissolving (or redissolving) the dry powder preparation (or the freeze-dried powder preparation) to obtain a liquid preparation.

In some embodiments, the pharmaceutically acceptable carrier is selected from, but not limited to: fillers, stabilizers (e.g., trehalose, glycine), flavoring agents (e.g., xylitol), disintegrating agents (e.g., sodium carboxymethylcellulose), binders (e.g., gelatin), lubricants (e.g., magnesium stearate).

In some embodiments, the stabilizing agent is selected from one or a combination of: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose and glucan.

In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharide, saccharin sodium, aspartame, sorbitol, xylitol and mannitol.

In some embodiments, the binder is selected from one or a combination of: sodium carboxymethylcellulose, hypromellose, and gelatin.

In some embodiments, the lubricant is selected from one or a combination of: comprises talcum powder, magnesium stearate and superfine silica powder.

In some specific embodiments, pharmaceutically acceptable carriers suitable for use in the present disclosure may also be mentioned, such as, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, a propellant, a humectant, a solvent, a solubilizer, an emulsifier, an antioxidant, a pH regulator and a preservative. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sucrose anhydride tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, Triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

In a fourth aspect, the present disclosure provides a method for preventing or treating chronic cervicitis with high risk HPV infection in a subject, comprising the steps of: administering to the subject a therapeutically effective amount of a nocardia rubra cell wall skeleton or pharmaceutical composition of the present disclosure.

"administering," "providing," "treating," when applied to an animal, human, cell, tissue, organ, or biological sample, refers to contacting a drug or medical device with the animal, human, cell, tissue, organ, or biological sample.

By "treating" is meant administering an internal or external drug (therapeutic agent, active ingredient or composition) (e.g., exosomes or pharmaceutical compositions of the present disclosure) or medical device to a subject who has been, suspected of having, or is susceptible to one or more diseases or symptoms thereof, in a subject (or population) being treated to alleviate (reduce, delay, ameliorate, cure) one or more symptoms of the disease, so as to achieve a clinically measurable degree.

The amount of drug (therapeutic agent, active ingredient or composition) that is effective to alleviate any symptoms of the disease is referred to as a therapeutically effective amount. May vary depending on a number of factors, such as the disease state, age and weight of the subject. It is understood that a drug (therapeutic agent, active ingredient or composition) may not be effective in alleviating a target disease or symptom thereof in an individual subject, but the drug (therapeutic agent, active ingredient or composition) is statistically effective against the target disease or symptom thereof as determined by any statistical test method known in the art, such as Student's T test, chi-square test, U-test by Mann and Whitney.

In some specific embodiments, the subject is an animal other than a human, e.g., a farm animal, a pet, a work animal, an ornamental animal, a production animal, a laboratory animal (e.g., rat, mouse, guinea pig, rabbit, dog, primate).

In some specific embodiments, the subject is a human. In some specific embodiments, the subject is suspected of having, diagnosed with, has had, or is susceptible to the disease of interest or symptoms thereof.

In some embodiments, the administration is 1-3 times a day, or once every two days. Different dosages are adopted according to different areas and degrees of the focus of the patient.

In some embodiments, the administration cycle lasts from 2 days to 6 months, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, or longer, and ranges between any two of the foregoing.

The same or a different pharmaceutically active ingredient may be administered at once or may be divided into a number of smaller unit doses to be administered at intervals. It will be understood that the exact dose, duration, and interval of treatment is a function of the disease being treated and can be determined using animal or clinical trial data inferences. The administration may comprise a single administration, or two or more administrations separated by a suitable time interval. Wherein two consecutive administrations are separated by 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half of a day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

"optional" means that the subsequently described events thereof can occur, but need not occur; as the case may be. For example, "optionally, portioned" means that product is allowed to be portioned, but is not necessarily.

The terms "a", "an", "the", and "the" include plural references unless expressly specified otherwise.

When referring to a range of values (e.g., 60 μ g to 120 μ g), this is intended to be a shorthand way of referring explicitly to each value falling within the range, including both fractional and integer values.

Detailed Description

The present disclosure is further described below in conjunction with the examples. These examples are not intended to limit the scope of the present disclosure. When the specific conditions are not specified, the operation is carried out under the conventional conditions, as recommended by the raw material supplier. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Examples

Example 1 commercially available Nocardia rubra cell wall skeleton

The red nocardia rubra cell wall skeleton is purchased from Liaoning Graishite biological pharmacy, Inc., Chinese medicine standard character S20030009 (the solid content of the cell wall skeleton in each cell should be not less than 60 mug, wherein the muramic acid content is not less than 1.0 mug, the sugar content is not less than 4.0 mug, and the re-dissolving volume is 2.0 ml).

Example 2 preparation of cell wall skeleton of Nocardia erythraea

The preparation method of the cell wall skeleton of the Chinese medicine standard character S20030009 is basically not obviously different from the following steps, but can be adjusted due to different production scales.

Thus, as an alternative, the following steps may be used to prepare the cell wall skeleton:

1. the cells were cultured according to a known method and collected. The cells are disrupted (e.g., by sonication or high pressure homogenizer). It is also permissible to crush the cells by any suitable method known in the art. The broken condition is checked under a microscope, the number of the visible bacteria in each visual field is not more than 5, and the standard is met by checking a plurality of visual fields (10 to 30) to be qualified.

2. Removing nucleic acid: the disrupted supernatant was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acids according to the procedures recommended by the enzyme supplier.

3. Removing protein: the precipitate is added with a common protease (e.g. trypsin) and the protein is removed according to the procedures recommended by the supplier of the enzyme.

4. Removing lipid: adding organic reagent (such as one or combination of acetone, diethyl ether, and ethanol) into the precipitate, and removing lipid according to conventional operation in the art.

5. Removing cell membranes: TritonX-100 was added to the precipitate, and the precipitate was collected by centrifugation and rinsed with PBS.

It should be understood that, between the above steps of removing impurities, the skilled person can adjust the order so as to make the steps compatible. After removing the non-cell wall components, the precipitate was redissolved in water for injection for use. Optionally, it can be sterilized at 115 deg.C for 20-30 minutes as a stock solution of cell wall skeleton.

Example 3 an exemplary method for preparing a pharmaceutical composition

1. The product obtained in example 1 or 2 is coated on a dressing (e.g., sterile gauze) to prepare a drug for external use.

2. The product obtained in example 2 was made into lyophilized powder.

3. Lotion preparation methods well known in the art may also be employed, for example: the lotion mostly takes water and alcohol as dispersion media; is prepared from active component, electrolyte, isoosmotic regulator, etc. in dispersing medium.

4. The product obtained in example 1 or 2 was prepared into capsules.

5. The product obtained in example 1 or 2 is dissolved in water for injection to prepare injection.

6. The product obtained in example 1 or 2 was prepared into pessaries.

Examples of effects

To a subject clinically diagnosed with chronic cervicitis, the cell wall skeleton of example 1 was administered. The results are shown in Table 1.

TABLE 1

HPV (-) means high risk HPV types tested negative.

Claims (4)

1. Use of a nocardia rubra cell wall scaffold in the preparation of a medicament, wherein:

the medicament is used for preventing or treating chronic cervicitis with high-risk HPV infection in a subject;

the high risk HPV is selected from any one or combination of the following: 16. types 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68;

the chronic cervicitis is selected from any one or a combination of the following: cervical columnar ectopy, cervical polyp, cervical mucositis, cyst of cervical gland, and cervical hypertrophy.

2. The use according to claim 1, the medicament being prepared in a dosage form selected from any one of: ointment, cream, suppository, emulsion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, film, patch, and injection.

3. The use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is a nocardia rubra cell wall skeleton corresponding to the national drug standard S20030009 or equivalent thereof.

4. Use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is obtained by a method comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the red nocardia to obtain a crushed product;

3.1) removing lipids from the disrupted product;

3.2) removing nucleic acids from the disruption product;

3.3) removing proteins from the disruption product;

3.4) removing cell membranes from the disruption product;

3.5) obtaining a red nocardia cell wall skeleton;

4) optionally, subpackaging;

5) optionally, freeze-drying the nocardia rubra cell wall skeleton;

wherein the content of the first and second substances,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or in parallel,

step 4) and step 5) can be interchanged;

the average particle size of the fractures is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500 nm.